沙门菌属pad蛋白的原核表达及抗原性的鉴定

Prokaryotic expression of Salmonella possible outer membrane adhesin and identification of its antigenicity

目的在大肠杆菌中表达沙门菌属pad蛋白,纯化后制备兔抗pad抗体。方法利用PCR方法从肠炎沙门菌中扩增出pad基因,构建pET-32a(+)-pad重组表达质粒;将重组表达质粒转化至大肠杆菌BL21(DE3)中,筛选阳性克隆,测序鉴定后,优化IPTG浓度、诱导温度和诱导时间以诱导重组蛋白的表达;以纯化的pad蛋白免疫家兔,制备抗pad多克隆抗体并进行鉴定。结果与结论成功扩增得到pad基因,经双酶切和测序证实重组表达质粒构建成功,经过诱导条件的优化,在大肠杆菌中表达出相对分子质量为41×103的目的蛋白;纯化的重组蛋白免疫家兔后,能够有效地刺激家兔产生特异性抗体,经琼脂糖双向扩散测定抗血清效价达到1:32以上。为进一步研制沙门菌属体外快速检测试剂打下了基础。

Objective To obtain Salmonella protein pad expressed in E. coli BL21 （DE3） to produce its polyclonal antibody. Methods The pad gene amplified from S. enteritidis by PCR was inserted into expression plasmid pET-32a（ ＋ ） to construct recombinant plasmid pET-32a（ ＋ ）-pad,which was then transformed into E. coli BL21 （DE3） for the expression pad under IPTG induction. The protein pad was purified with Ni ~ -NTA system and used to immunize rabbits to prepare the anti-pad antibody, whose properties were identified. Results The Salmonella pad gene was confirmed by DNA sequencing, and positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. After induc- tion with IFFG, pad with Mr being 41 x 103 was expressed in E. coli B121 （ DE3 ） and purified. Conclusion The recombi- nant expression plasmid of pad is constructed successfully and expressed in E. coli BI21 （ DE3 ）. The prepared rabbit anti- pad antibody has a high titer, making easy the development of rapid detection of Salmonella in the future.